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Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation

Oncogene volume 33, pages 2610–2619 (2014)Cite this article

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Abstract

Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E-cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation.

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Abbreviations

AnxA1:

Annexin A1

AnxA2:

Annexin A2

ER:

estrogen receptor

EGF:

epidermal growth factor

EGFR:

epidermal growth factor receptor

GFP:

green fluorescent protein

RNAi:

RNA interference

shRNA:

small hairpin RNA

TNBC:

triple negative breast cancer

WT:

wild type

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Acknowledgements

We thank Dr Erik Danen and Dr Leo Price for critically reading the manuscript. This work was supported by grants from the Dutch Cancer Society (KWF; grant UL 2006-3538 and UL 2007-3860), EU FP7-Metafight (grant no 201862), EUFP7 Systems Microscopy NoE (258068), TI Pharma project T3-107, the Dutch Organization for Scientific Research (NWO 911-02-022), Horizon Breakthrough (NWO 93518003) and Centre for Biomedical Genetics.

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Author notes

  1. L Cao and L Winkel: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Toxicology, LACDR, Leiden University, Leiden, The Netherlands

    M de Graauw, S E le Dévédec, M Klop, C Pont, V-M Rogkoti, A Tijsma, A Chaudhuri, R Lalai, L Price & B van de Water

  2. Imaging and Bio-informatics group, LIACS, Leiden University, Leiden, The Netherlands

    L Cao, K Yan & F Verbeek

  3. Biomechanics Laboratory, Erasmus MC, Rotterdam, The Netherlands

    L Winkel

  4. Department of Molecular Pathology, Netherland Cancer Institute, Amsterdam, The Netherlands

    M H A M van Miltenburg

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  1. M de Graauw
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Correspondence to M de Graauw or B van de Water.

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de Graauw, M., Cao, L., Winkel, L. et al. Annexin A2 depletion delays EGFR endocytic trafficking via cofilin activation and enhances EGFR signaling and metastasis formation. Oncogene 33, 2610–2619 (2014). https://doi.org/10.1038/onc.2013.219

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